Arabidopsis SBT5.2 and SBT1.7 subtilases mediate C-terminal cleavage of flg22 epitope from bacterial flagellin.

Plants initiate specific defense responses by recognizing conserved epitope peptides within the flagellin proteins derived from bacteria. Proteolytic cleavage of epitope peptides from flagellin by plant apoplastic proteases is thought to be crucial for the perception of the epitope by the plant receptor. However, the identity of the plant proteases involved in this process remains unknown. Here, we establish an efficient identification system for the target proteases in Arabidopsis apoplastic fluid; the method employs native two-dimensional electrophoresis followed by an in-gel proteolytic assay using a fluorescence-quenching peptide substrate. We designed a substrate to specifically detect proteolytic activity at the C-terminus of the flg22 epitope in flagellin and identified two plant subtilases, SBT5.2 and SBT1.7, as specific proteases responsible for the C-terminal cleavage of flg22. In the apoplastic fluid of Arabidopsis mutant plants deficient in these two proteases, we observe a decrease in the C-terminal cleavage of the flg22 domain from flagellin, leading to a decrease in the efficiency of flg22 epitope liberation. Consequently, defensive reactive oxygen species (ROS) production is delayed in sbt5.2 sbt1.7 double-mutant leaf disks compared to wild type following flagellin exposure.

A Crucial Role for the Small GTPase Rac1 Downstream of the Protein Kinase Akt2 in Insulin Signaling that Regulates Glucose Uptake in Mouse Adipocytes.

Insulin-stimulated glucose uptake is mediated by translocation of the glucose transporter GLUT4 to the plasma membrane in adipocytes and skeletal muscle cells. In both types of cells, phosphoinositide 3-kinase and the protein kinase Akt2 have been implicated as critical regulators. In skeletal muscle, the small GTPase Rac1 plays an important role downstream of Akt2 in the regulation of insulin-stimulated glucose uptake. However, the role for Rac1 in adipocytes remains controversial. Here, we show that Rac1 is required for insulin-dependent GLUT4 translocation also in adipocytes. A Rac1-specific inhibitor almost completely suppressed GLUT4 translocation induced by insulin or a constitutively activated mutant of phosphoinositide 3-kinase or Akt2. Constitutively activated Rac1 also enhanced GLUT4 translocation. Insulin-induced, but not constitutively activated Rac1-induced, GLUT4 translocation was abrogated by inhibition of phosphoinositide 3-kinase or Akt2. On the other hand, constitutively activated Akt2 caused Rac1 activation, and insulin-induced Rac1 activation was suppressed by an Akt2-specific inhibitor. Moreover, GLUT4 translocation induced by a constitutively activated mutant of Akt2 or Rac1 was diminished by knockdown of another small GTPase RalA. RalA was activated by a constitutively activated mutant of Akt2 or Rac1, and insulin-induced RalA activation was suppressed by an Akt2- or Rac1-specific inhibitor. Collectively, these results suggest that Rac1 plays an important role in the regulation of insulin-dependent GLUT4 translocation downstream of Akt2, leading to RalA activation in adipocytes.

One-Step Synthesis of One-Dimensional Supramolecular Assemblies Composed of Helical Macromolecular Building Blocks.

Living systems achieve sophisticated functions using supramolecular protein assemblies, in which the protein building blocks possess a specific secondary structure and are noncovalently arranged in a preprogrammed manner. Herein, we demonstrate the one-step synthesis of one-dimensional macromolecular assemblies by simply mixing a glycine-based isocyanide with a nickel catalyst, in which helical constituent polymers are linked end-to-end through multiple hydrogen bonds. The applicable scope of this approach is not confined to a particular monomer bearing a specially designed pendant, but covers a wide range of glycine-based isocyanides with or without aromatic and other functional groups. Surprisingly, copolymerization with an analogous chiral isocyanide (1 mol %) afforded an almost perfect one-handed helical supramolecular fiber owing to intramolecular/intermolecular dual chiral amplifications. The simplicity and broad applicability of this approach, which can also afford exquisite chiral amplification, enable the creation of a wide variety of functional supramolecular assemblies and provide access to new supramolecular materials.

Analysing the evolutional and functional differentiation of four types of Daphnia magna cryptochrome in Drosophila circadian clock.

Cryptochrome (CRY) plays an important role in the input of circadian clocks in various species, but gene copies in each species are evolutionarily divergent. Type I CRYs function as a photoreceptor molecule in the central clock, whereas type II CRYs directly regulate the transcriptional activity of clock proteins. Functions of other types of animal CRYs in the molecular clock remain unknown. The water flea Daphnia magna contains four Cry genes. However, it is still difficult to analyse these four genes. In this study, we took advantage of powerful genetic resources available from Drosophila to investigate evolutionary and functional differentiation of CRY proteins between the two species. We report differences in subcellular localisation of each D. magna CRY protein when expressed in the Drosophila clock neuron. Circadian rhythm behavioural experiments revealed that D. magna CRYs are not functionally conserved in the Drosophila molecular clock. These findings provide a new perspective on the evolutionary conservation of CRY, as functions of the four D. magna CRY proteins have diverse subcellular localisation levels. Furthermore, molecular clocks of D. magna have been evolutionarily differentiated from those of Drosophila. This study highlights the extensive functional diversity existing among species in their complement of Cry genes.

Selective oxidation of B800 bacteriochlorophyll a in photosynthetic light-harvesting protein LH2.

Engineering chlorophyll (Chl) pigments that are bound to photosynthetic light-harvesting proteins is one promising strategy to regulate spectral coverage for photon capture and to improve the photosynthetic efficiency of these proteins. Conversion from the bacteriochlorophyll (BChl) skeleton (7,8,17,18-tetrahydroporphyrin) to the Chl skeleton (17,18-dihydroporphyrin) produces the most drastic change of the spectral range of absorption by light-harvesting proteins. We demonstrated in situ selective oxidation of B800 BChl a in light-harvesting protein LH2 from a purple bacterium Rhodoblastus acidophilus by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone. The newly formed pigment, 3-acetyl Chl a, interacted with the LH2 polypeptides in the same manner as native B800. B850 BChl a was not oxidized in this reaction. CD spectroscopy indicated that the B850 orientation and the content of the α-helices were unchanged by the B800 oxidation. The nonameric circular arrangement of the oxidized LH2 protein was visualized by AFM; its diameter was almost the same as that of native LH2. The in situ oxidation of B800 BChl a in LH2 protein with no structural change will be useful not only for manipulation of the photofunctional properties of photosynthetic pigment-protein complexes but also for understanding the substitution of BChl to Chl pigments in the evolution from bacterial to oxygenic photosynthesis.

MicroRNA-342 inhibits tumor growth via targeting chemokine CXCL12 involved in macrophages recruitment/activation.

MicroRNAs (miRNAs) play important roles in initiation, development, progression and metastasis of tumors. MiR-342 has been reported as a tumor suppressor or an onco-miRNA based on functions or expression changes in various types of cancers. However, the biological roles and underlying molecular mechanisms of miR-342 in tumorigenesis remain largely unknown. Here, we found that miR-342 was expressed significantly less in a murine MS-K tumor cell line that showed riched blood vessels. Over-expression of miR-342 in MS-K cells inhibited cell proliferation, colony formation, reduced frequency of S phase population in vitro and suppressed tumor growth in vivo. Moreover, increasing miR-342 impeded blood vessels formation and accumulation of macrophages (CD11b+ ) in tumors. By bioinformatic analysis and dual-luciferase reporter assays, chemokine CXCL12 was identified as a direct target of miR-342. Restored Cxcl12 expression in MS-K-miR-342 cells could rescue cell proliferation in vitro. In MS-K-miR-342 tumor-infiltrated macrophages, expression of proangiogenic genes (Vegf-A and Thbs1) and M2-subtype macrophage markers (Cd163, Dectin1 and Ym1) was significantly down-regulated compared with controls. Moreover, lower level of Cxcl12 and its receptor Cxcr4 was observed in the macrophages of MS-K-miR-342 tumors, and MS-K-miR-342 derived miR-342, but not endogenous miR-342, might contribute to Cxcl12 suppression in TAM. These results suggest that miR-342 is involved in MS-K tumor growth as a tumor suppressor by targeting chemokine CXCL12.

Selective Removal of B800 Bacteriochlorophyll a from Light-Harvesting Complex 2 of the Purple Photosynthetic Bacterium Phaeospirillum molischianum.

The selective removal of B800 bacteriochlorophyll (BChl) a from light-harvesting complex 2 (LH2) in purple photosynthetic bacteria is a clue about elucidation of the mechanism for the transfer of energy from these pigments to B850 BChl a and their roles in the LH2 protein structure. We demonstrated that the kinetics of the removal of B800 BChl a from two representative LH2 proteins derived from Phaeospirillum molischianum and Rhodoblastus acidophilus differed significantly, in contrast to the calculated binding enthalpy. These results may be interpreted as changes in the local structure near B800 BChl a with respect to the geometries of the original crystal structures upon removal of B800 BChl a. Despite the difficulty of removing B800 BChl a from molischianum-LH2, we prepared the molischianum-LH2 protein lacking B800 BChl a by combination of two detergents, n-dodecyl ß-d-maltoside and n-octyl ß-d-glucoside, under acidic conditions. Spectral and atomic force microscopy analyses indicated that the absence of B800 BChl a had little effect on the local structure in the vicinity of B850 BChl a and the circular arrangement in this protein. These results suggest that the hydrophobic domain near B850 BChl a is rigid and plays a major role in the structural formation of molischianum-LH2.

Self-assembled monolayers of sulfonate-terminated alkanethiols investigated by frequency modulation atomic force microscopy in liquid.

A molecular-scale understanding of self-assembled monolayers (SAMs) of sulfonate-terminated alkanethiols is crucial for interfacial studies of functionalized SAMs and their various applications. However, such an understanding has been difficult to achieve because of the lack of direct information on these molecular-scale structures in real space. In this study, we investigated the structures of sulfonate SAMs of sodium 11-mercapto-1-undecanesulfonate (MUS) by frequency modulation atomic force microscopy (FM-AFM) in liquid. The subnanometer-resolution FM-AFM images showed that the single-component MUS SAM prepared in pure water had random surface structures. In contrast, the MUS SAM prepared in a water-ethanol mixed solvent showed periodic striped structures with a flat-lying conformation. The results suggest a significant solvent effect on molecular-scale structures of long-chain sulfonate SAMs. In addition, we investigated the molecular-scale structures of mixed SAMs of MUS and 11-mercapto-1-undecanol (MUO) with alkane chains of the same length. The FM-AFM images of the mixed SAMs showed clear phase separation between MUS SAM and MUO SAM domains. In the MUO SAM domains, the incorporated MUS molecules appeared as protrusions. The results obtained in this study provide direct structural information on long-chain sulfonate and mixed SAMs.

CCL11-induced eosinophils inhibit the formation of blood vessels and cause tumor necrosis.

We previously demonstrated that IL-18 and CCL11 were highly expressed in an NFSA tumor cell line that showed limited angiogenesis and severe necrosis. However, IL-18 was not responsible for the immune cell accumulation and necrosis. Here, we attempted to clarify the relevance of CCL11 in angiogenesis and tumor formation. We established CCL11-overexpressing MS-K cell clones (MS-K-CCL11) to assess the role of CCL11 in immune cell accumulation and angiogenesis. The MS-K-CCL11 cells did not form tumors in mice. MS-K-CCL11-conditioned medium (CM) and recombinant CCL11 induced macrophage and eosinophil differentiation from bone marrow cells. The MS-K-CCL11-CM effectively recruited the differentiated eosinophils. Furthermore, the eosinophils damaged the MS-K, NFSA and endothelial cells in a dose-dependent manner. Administration of an antagonist of CCR3, a CCL11 receptor, to NFSA tumor-bearing mice restored the blood vessel formation and blocked the eosinophil infiltration into the NFSA tumors. Furthermore, other CCL11-overexpressing LM8 clones were established, and their tumor formation ability was reduced compared to the parental LM8 cells, accompanied by increased eosinophil infiltration, blockade of angiogenesis and necrosis. These results indicate that CCL11 was responsible for the limited angiogenesis and necrosis by inducing and attracting eosinophils in the tumors.

Inhibition of blood vessel formation in tumors by IL-18-polarized M1 macrophages.

We previously showed that interleukin (IL)-18 produced by NFSA cells induced the M1 type of macrophages in NFSA tumors, caused the destruction of endothelial cells in vitro and may have resulted in the necrosis of NFSA tumors by enhancing macrophage phagocytosis and cytotoxicity. However, the effect of IL-18 on blood vessel formation in vivo has not been elucidated. MS-K cells do not express il-18, and they form tumors with well-developed blood vessels. Here, we established IL-18-over-expressing MS-K cell clones (MS-K-IL-18) to address the roles of IL-18 in angiogenesis. The over-expression of IL-18 inhibited the proliferation rate of the MS-K-IL-18 cells in vitro and blood vessel formation in the MS-K-IL-18 tumors. Interestingly, CD14-positive cells from the MS-K-IL-18 tumor had up-regulated expression of the M1-type macrophage marker il-6 and down-regulated expression of interferon (ifn)-γ. Furthermore, FACS analysis showed more accumulation of CD11b+/CD80+ M1 macrophages in the MS-K-IL-18 tumors than in the parental MS-K tumor. Moreover, an in vitro coculture assay showed that MS-K-IL-18-conditioned medium (CM) stimulated macrophages to induce the apoptosis of endothelial cells. Cumulatively, our data showed that IL-18 inhibited tumor blood vessel formation in vivo.